Thrombotic occlusion of the arteries of the heart or brain kills and disables 12.7 million people a year worldwide. Therapeutic dissolution of these thrombi by plasmin, an enzyme that digests fibrin, reduces death and disability from heart attacks and strokes. Plasmin is generated from the zymogen plasminogen (Pg) by Pg activators such as streptokinase (SK) and tissue Pg activator (TPA). Plasmin generation and activity is tightly regulated by protein interactions with inhibitors (e.g., 12-antiplasmin), substrates (e.g., fibrin) and other molecules. Safer and more effective agents for dissolving thrombi would markedly reduce mortality and disability in patients with cardiovascular disease. Our long term goal is to help elucidate the protein-protein interactions that regulate the activity of the P(g) system and to translate these insights into potential therapeutics. During the last funding period of this grant we have helped to delineate the elegant interactions through which SK converts Pg (without cleavage) into the most catalytically efficient Pg activator. Insights have been made into defining: 1) the mechanisms through which SK initiates Pg activation in the absence of fibrin; 2) the structural elements in SK that can be altered to produce extraordinarily potent and fibrin-specific Pg activators; 3) the basis for molecular complementarities between SK and Pg that mediates species-specific Pg activation and, 4) how a novel, small, molecule can be altered to form a complex that efficiently activates human Pg. The mechanistic insights from these studies have lead to the discovery of Pg activators that work through mechanisms different from SK to activate Pg with exquisite specificity in vitro and in vivo. This continuation proposal seeks to characterize these novel molecules and define their potential therapeutic properties in relevant in vivo models of thrombosis and stroke. In particular we seek to: 1) reprogram the mechanisms of action and the fibrinolytic potential of a new Pg activator; 2) define which novel Pg activators display greater fibrin-specificity and fibrinolytic potency in vivo in humanized thrombosis models and, 3) examine a central tenet of the fibrinolytic field by determining whether fibrin-specific Pg activation decreases the risk of cerebral hemorrhage in vivo. Continued analysis of these unique mechanisms of Pg activation will further enhance our understanding of how the catalytic activity and specificity of P(g) system is regulated and, will define whether these novel Pg activators, with tailored molecular modifications, have potential for improving the treatment for patients with thrombosis. PUBLIC HEALTH RELEVANCE: Clots that block the flow of blood are the primary causes of stroke and heart attacks. Finding safer and more effective ways to dissolve blood clots could save millions of lives. We have discovered novel clot dissolving molecules and, in this project we seek to determine how effective and safe they are in models of stroke, etc.